Human papillomaviruses (HPV) cause very common infections of men, women and children. These are of particular medical importance and scientific significance since a subset of HPVs cause cancers of cervix, oropharynx, and anogenital region. In this grant proposal we investigate the functions of the E6 protein in HPV type 16, the most prevalent HPV associated with malignant progression. Aim 1 is a comprehensive investigation of the molecular pathways and interacting partners of E6 that are essential for keratinocyte immortalization. Cancer associated HPV E6 proteins bind and degrade the tumor suppressor p53 and stimulate expression of the catalytic component of telomerase hTERT. HPV-16 E6 also binds to other cellular proteins, but the significance of these interactions is largely unknown. We reported that the E6 interacting protein Ada3, which binds p53 and histone acetyltransferases, mediates p53 acetylation and induction of cell senescence. Recent results demonstrate an additional role for E6 and p53 acetylation in autophagic cell death. E6 is also necessary for stable viral genome replication and in Aim 2, we explore the functions of E6 that are necessary for viral replication in monolayer culture in which the genome is maintained as low copy episomes and in differentiated keratinocyte models that reproduce the stage of viral genome amplification. In this grant proposal, we combine the use of well-characterized HPV-16 E6 mutations, RNA silencing, and expression of dominant negative cellular factors to decipher their functions in human keratinocyte immortalization and HPV genome replication. This research project has already produced novel insights into the molecular pathways that govern p53 activity, escape from replicative senescence, and neoplastic progression and will result in new understandings of the contributions of E6 to replication of episomal HPV genomes and potential targets for HPV E6 directed therapeutics.